This application relates to a new mutants of the enzyme dihydrofolate reductase, and to the use of these mutants as selectable markers and for gene therapy to produce drug resistant bone marrow or peripheral stem cells.
Dihydrofolate reductase (DHFR, 5,6,7,8-tetrahydrofolate:NADP+oxidoreductase, EC 1.5.1.3) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, an essential carrier of one-carbon units in the biosynthesis of thymidylate, purine nucleotides, serine and methyl compounds. DHFR is an essential enzyme in both eukaryotes and prokaryotes.
In rapidly dividing cells, the inhibition of DHFR results in the depletion of cellular tetrahydrofolates, inhibition of DNA synthesis and cell death. Because of this, folate analogs which inhibit DHFR, for example methotrexate (MTX), are used as antineoplastic agents. The utility of “antifolate” treatments of this type is limited by two factors. First, tumor tissues may rapidly develop resistance to the antifolate, rendering the treatment ineffective. Second, the treatment may be toxic to rapidly dividing normal tissues, particularly to bone marrow or peripheral stem cells.
International Patent Publication No. WO94/24277, which is incorporated herein by reference, discloses mutant forms of human DHFR which have increased resistance to inhibition by antifolates used in therapy including MTX. The specific mutants disclosed differ from wild-type human DHFR as a result of a single mutation occurring at amino acid 15. 31 or 34.
Mutations at amino acid 22 of human DHFR have also been shown to reduce the sensitivity of the enzyme to antifolate inhibition. Ercikan et al., in Chemistry and Biology of Pieridines and Folates, J. E. Ayling, ed. Plenum Press (1993). In these mutants, the amino acids isoleucine, methionine, phenylalanine and tyrosine are substituted for the leucine of the wild-type enzyme.